Two groups of proteins are characterized by specific binding to vitamin B12, (cobalamin), hereafter called cobalamin binding proteins (CBP). One group consists of proteins involved in the assimilation of cobalamin and the other group is involved in the metabolism and utilization of cobalamin and includes the enzymes using cobalamin as a cofactor. Human assimilation of dietary cobalamin (Cbl) is a complex process with three successive Cbl-transporters involved: haptocorrin (HC), intrinsic factor (IF) and transcobalamin (TC) (1-4). These proteins strongly bind to Cbl which is synthesized by bacteria. The three CBPs provide uptake of the Cbl from the food to the body. IF is the main binder of Cbl in the intestinal tract and the IF-Cbl complex binds to an intestinal receptor resulting in the internalization of the Cbl to the blood (3,4). In the blood the TC-Cbl complex distributes assimilated Cbl among the tissues where it binds to one or more specific receptors present in the cell membrane (2,4). A significant amount of Cbl circulates in blood bound to HC (1,4). The exact function of HC is not identified. Inherited HC deficiency does not provoke any visible pathological effect (5) in contrast to the cases of IF- and TC-deficiencies (6,7).
Two Cbl dependent enzymatic reactions have been identified in mammals. The conversion of methylmalonyl-CoA to succinyl-CoA involves adenosyl-Cbl (Ado-Cbl) as a cofactor. Methyl-Cbl is a cofactor in the synthesis of methionine from homocysteine. Prior to the present invention, only small amounts of CBP have been isolated. Purification of Cbl binders is complicated by low concentration of these proteins in natural sources (1-4). Isolation of 1 mg of TC requires, for instance, 150-300 liters of human plasma (8,9). Human IF can be isolated from stomach juice which contains about 1 mg IF per liter. Isolation of pure TC and IF from natural sources is also complicated by the presence of relatively large amounts of HC in these sources. An expression system has been established for IF and TC in insect cells (10,11) and recombinant proteins were obtained at the level of 10-100 yg. We have expressed human and bovine TC in yeast and obtained 5-7 mg recombinant protein from a total of 1 liter of fermentation media (12). Human IF has been expressed in yeast and the yield was in the range of 1-4 mg per liter of total fermentation media (13). These expression systems are expensive to use and they deliver a mixture of holo- and apo-forms of the CBP. This is also the case for natural sources. The holo-form is the CBP in complex with Cbl, whereas the apo form is a CBP not complexed to Cbl. For use in diagnostic kits and other analytical purposes it is important to achieve purely either the apo- or holo-form of CBP.
IF isolated from human gastric juice is used for diagnostic purposes in the Schilling test where the patient ingests the isolated IF. Therefore, there is a risk for transmission of human diseases from the donor to the patient. Similarly, the use of IF isolated from pig or recombinant human IF isolated from the yeast expression media which contains an enzymatic hydrolysate of meat, may cause a risk for transmission of animal diseases to the patient. The use of recombinant human IF from a plant expression system will eliminate these problems.
Plants do not contain Cbl or CBPs. Therefore, recombinant plants with an inserted gene for i.e. human IF will express only the apo-form of IF. This makes plants very suitable as a “factory” for production of the apo-form of CBPs with no contamination of the holo-form. In other eukaryotic expression systems the recombinant CBP will be more or less saturated with Cbl resulting in a mixture of holo- and apo-form CBP. All other eukaryotic organisms except plants use Cbl in enzymatic reactions and therefore contain Cbl. Similarly, the use of transgenic plants results in recombinant CBP free of other CBPs since plants do not naturally express CBP. Isolation of IF from other sources, i.e. gastric juice results in a preparation contaminated with some HC since both CBPs are present in gastric juice.
CBPs expressed in plants but not other organisms are only in the apo-form and therefore the plant expression system gives the opportunity for purification of the CBP by affinity column chromatography with a cobalamin column. The holo-form of CBP does not bind to the cobalamin column. Therefore, CBPs expressed in plants are easier to purify than CBPs expressed in other organisms.
CBPs expressed in plants can be designed to have or alternatively not to have identical amino acid sequences as their corresponding native proteins. Posttranslational modifications such as disulphide bond formation between cystein residues will be present in the recombinant proteins. For CBPs with amino acid sequences identical with native CBPs the localization of disulphide bonds are expected to be identical, since CBPs from plants binds Cbl. Therefore the tertiary structure of recombinant CBPs are expected to be identical to native CBPs. The specific binding of recombinant human IF (rhIF) from plants in a complex with Cbl to the intestinal receptor cubilin, support an identical tertiary structure between native hIF and rhIF from plant. The glycosylation of recombinant CBPs from plants will not be identical to the glycosylation of the corresponding native CPBs, since glycosylation is cell type specific. Therefore, rhIF from plants differ from native IF by the composition of the sugars in the carbohydrate chains.
The plant expression system will be very useful for large scale production of CBP, since the growth of recombinant plants is low-tech and furthermore, it is simple to scale up the production of recombinant protein by simply planting more land. The plant expression system gives the opportunity to use large amounts of recombinant CBP in therapeutic programs, e.g. elderly people with reduced uptake of vitamin B-12. Furthermore, the CBP may be used after no or very little purification since many plant species, useful for transgenic expression of proteins, are used for human consumption e.g. maize, potato, barley, rice, carrot.
A need exists for a better way to produce these CBPs.